PROJECT SUMMARY/ABSTRACT Biliary atresia is the most common cause of end-stage cirrhosis in children and the number one indication for pediatric liver transplantation. It results from an inflammatory and fibrosing obstruction of extrahepatic bile ducts that presents as neonatal jaundice. Despite prompt diagnosis and surgical treatment, the disease progresses and causes substantial morbidity and mortality. Although the etiology remains largely undefined, studies pursuing the previous aims of this award have advanced knowledge of pathogenic mechanisms of disease. Specifically, we uncovered a sequential activation of the innate and adaptive immune systems, with a prominent type-1 inflammatory response that targets the cholangiocytes and promotes epithelial injury. Consistent with a multifactorial basis of disease, we also found type-2 signals and directly linked them to a novel paracrine circuit involving lineage-negative (Linneg) cells functionally committed to type-2 innate lymphoid cells (ILC2s) that secrete soluble mitogenic factor(s) to neighboring cholangiocytes. In this competing renewal application, we provide substantial preliminary data that forms the rationale for a unifying hypothesis that novel immature immune cells in the liver and bile ducts have dual roles as regulators of the immune response and the source of survival signals. This hypothesis will be tested in three closely related but independent aims. In Aim 1, we will investigate how ILC2s use amphiregulin (Areg) to promote epithelial repair in bile ducts. This will be done by validating initial findings that Areg induces cholangiocyte proliferation in vivo, the molecular mechanisms of growth, and how Areg signaling protects neonatal mice against the virus-induced phenotype of biliary atresia. In Aim 2, we will define the tissue population by Linneg cells and their roles in epithelial proliferation. This aim builds on initial experiments using single-cell sequencing strategies that show the functional commitment of Linneg cells based on their population of the hepatic or biliary environments. Following validation of these cells using complementary technologies, we will directly examine how novel bile duct- resident BIM cells and their expression of IL-6 regulate cholangiocyte proliferation and epithelial injury in experimental biliary atresia. And in Aim 3, we will determine how Linneg CR71+ cells that exist in extramedullary hematopoietic cells that reside in the neonatal liver promote biliary atresia. Based on preliminary data, we propose studies to explore their role as a susceptibility factor for experimental biliary atresia, and to investigate the mechanisms they use to quench the inflammatory response of neonatal myeloid and NK cells. Upon completion, the proposed experiments will advance our understanding of the pathogenic mechanisms of disease and uncover new cell groups and molecular signals that promote repair of the injured tissue, thus identifying potential targets for new therapies to block progression of disease and foster long-term survival of patients with biliary atresia.